Glycomics, extracellular matrix, and anti-glycan antibodies in exfoliation syndrome.

Exfoliation syndrome (XFS) is considered to be a disease of extracellular matrix. Here we review key experimental evidence of aberrations in structure, expression, and function of glycoproteins, complex carbohydrates, and glycosaminoglycans found in extracellular matrix components forming exfoliation material in patients presenting with XFS. We hypothesize that certain components of the accumulating exfoliation material can become immunogenic, and multiple natural antibodies or autoantibodies are generated. Anti-glycan antibodies (AGAs) can be captured on Printed Glycan Array. Our preliminary results show robust immunoprofiles of AGAs in sera of patients with XFS, and the significant presence of AGAs in aqueous humor of these patients. These findings offer insight into the dynamics of AGAs during the development of XFS that could lead to the identification of the AGA-based XFS immuno-signature.

Immune phenomena in glaucoma and conformational disorders: why is the second eye not involved?

Data along several lines of evidence have suggested that a systemic autoimmune response may be provoked in glaucoma and could contribute to retinal ganglion cell loss. If such an autoimmune response exists, one could predict that in cases of unilateral glaucoma, autoantibodies generated would affect both eyes, leading to damage in the unaffected, contralateral eye in an intraocular pressure-independent manner. However, such an effect has not yet been reported. There are currently no data to reconcile these contrasting observations but a review of the literature suggests a possible explanation.

Gene therapy for primary immunodeficiencies: Part 2.

Gene therapy has become an attractive alternative therapeutic strategy to allogeneic transplant for primary immunodeficiencies (PIDs) owing to known genetic defects. Clinical trials using gammaretroviral vectors have demonstrated the proof of principle of gene therapy for Wiskott-Aldrich syndrome (WAS) and chronic granulomatous disease (CGD), but have also highlighted limitations of the technology. New strategies based on vectors that can achieve more robust correction with less risk of insertional mutagenesis are being developed. In this review we present the status of gene therapy for WAS and CGD, and discuss the emerging application of similar strategies to a broader range of PIDs, such as IPEX syndrome.

Serum antiphospholipid antibody levels in pseudoexfoliation.

PURPOSE: To evaluate the levels of serum antiphospholipid antibodies in patients with pseudoexfoliation syndrome (PXS), pseudoexfoliation glaucoma (PXG), primary open-angle glaucoma, and healthy controls. MATERIALS AND METHODS: This prospective study included 17 patients with PXS, 19 patients with pseudoexfoliation glaucoma, 15 patients with primary open-angle glaucoma, and 19 normal individuals. Venous blood samples were obtained in the morning after an overnight 8-hour fasting. Anticardiolipin antibodies, isotypes IgG and IgM were measured by means of an enzyme-linked immunosorbent assay. Lupus anticoagulant antibodies were measured by dilute Russell viper venom time screen test. RESULTS: Mean±standard mean of error of anticardiolipin antibody IgG levels in patients with PXS and PEG were significantly higher than those of the controls (P<0.05). The mean lupus anticoagulant antibody levels of the controls were not statistically different from the levels of patients with PXS, pseudoexfoliation glaucoma, and primary open-angle glaucoma (P>0.05). The anticardiolipin antibody IgG concentrations above the cutoff value of 15 GPL/mL were found in 8 patients (21.05%) with pseudoexfoliation. There was no individual in the control group having anticardiolipin antibody IgG level above the cutoff value (P<0.05). CONCLUSIONS: Elevated serum antiphospholipid antibodies, a risk factor for cardiovascular and cerebrovascular disease, is more common in patients with PXS and pseudoexfoliation glaucoma than in the healthy controls and in patients with primary open-angle glaucoma.

Conjunctival inflammatory cells and their predictive role for deep sclerectomy in primary open-angle glaucoma and exfoliation glaucoma.

PURPOSE: To investigate the conjunctival inflammatory alterations of patients with primary open-angle glaucoma (POAG) and exfoliation glaucoma (ExG) and correlate the findings with the success of deep sclerectomy (DS) surgery and with the patients' medical history. METHODS: Altogether 25 POAG and ExG patients of the prospective DS study were divided, based on the diagnosis and success of the operation, into 4 groups, POAG S (success), POAG F (failure), ExG S, and ExG F. Controls were obtained from other ophthalmologic surgery patients who did not have glaucoma, and their conjunctiva was examined to be normal. Inflammatory cell subtypes in the conjunctiva were identified and quantified by using immunohistochemistry and monoclonal antibodies: CD3 (T-lymphocyte marker), CD4 (T-helper lymphocyte marker), CD8 (T-cytotoxic lymphocyte marker), CD20 (pan-B cell marker), CD38 (plasma cell marker), CD45RA (naïve T-cell marker), and CD68 (macrophage marker). RESULTS: Higher numbers of inflammatory cells were found in the conjunctiva of the glaucoma patients on medical treatment compared with the normal conjunctiva of the controls. Moreover, T-lymphocytes, T-helper lymphocytes, T-cytotoxic lymphocytes, B cells, plasma cells, and macrophages were found in significantly higher numbers in patients in whom DS failed during the follow-up period of 2.5 years than those with surgical success. CONCLUSIONS: High numbers of cytotoxic and helper T-lymphocytes, plasma cells, and macrophages indicate a chronic inflammatory reaction in the conjunctiva of glaucoma patients. The chronic inflammation is most probably owing to the chronic topical treatment of the patients and seems to be a significant risk factor for DS surgery failure.

Proinflammatory cytokines are involved in the initiation of the abnormal matrix process in pseudoexfoliation syndrome/glaucoma.

Pseudoexfoliation (PEX) syndrome, which is an age-related, generalized elastotic matrix process, currently represents the most common identifiable risk factor for open-angle glaucoma. Dysregulated expression of proinflammatory cytokines has been implicated in the initiation of various fibrotic disorders and in the pathophysiology of glaucoma. Here we investigated the presence, expression, regulation, and functional significance of proinflammatory cytokines in eyes with early and late stages of PEX syndrome/glaucoma in comparison with normal and glaucomatous control eyes using multiplex bead analysis, immunoassays, real-time PCR, Western blotting, immunohistochemistry, and cell culture models. Early stages of PEX syndrome were characterized by approximately threefold (P < 0.005) elevated interleukin (IL)-6 and IL-8 levels in the aqueous humor and a concomitant approximately twofold (P < 0.001) increase in mRNA expression levels in anterior segment tissues as compared with controls. In contrast, late stages of PEX syndrome/glaucoma did not differ significantly from controls. IL-6, IL-6 receptor, and phospho-signal transducer and activator of transcription 3 could be mainly localized to walls of iris vessels and to the nonpigmented epithelium of ciliary processes. IL-6 and IL-8 were significantly up-regulated by ciliary epithelial cells in response to hypoxia or oxidative stress in vitro, whereas IL-6, but not IL-8, induced the expression of transforming growth factor-beta1 and elastic fiber proteins. These findings support a role for a stress-induced, spatially, and temporally restricted subclinical inflammation in the onset of the fibrotic matrix process characteristic of PEX syndrome/glaucoma.

Protein macroarray profiling of serum autoantibodies in pseudoexfoliation glaucoma.

PURPOSE: Complex repertoires of IgG autoantibodies have been detected against ocular antigens in patients with glaucoma. The goal was to identify and characterize the IgG autoantibody repertoires in sera of patients with pseudoexfoliation glaucoma (PXFG) with protein macroarrays. METHODS: Serum samples of 21 patients with PXFG and 19 age- and sex-matched control subjects were profiled on high-density colony protein macroarrays expressing His-tagged recombinant human proteins derived from a human fetal brain cDNA library. Statistically prevalent expression clones in the PXFG group were sequenced. mRNA expression of identified antigens was examined in the rat ganglion cell line RGC-5 and in human brain and optic nerve cDNA. The IgG immunoreactivity of the sera of 20 control and 26 PXFG patients to purified C6orf129 was analyzed in a reverse enzyme-linked immunosorbent assay. RESULTS: An increased prevalence was detected among the PXFG patients of serum antibodies to seven proteins: C6orf129; stathmin-like 4; transmembrane protein 9 domain family, member B; fibroblast growth factor receptor 3; cleft lip and palate transmembrane protein 1; EH-domain-containing protein 1; and eukaryotic translation elongation factor 2. All antigens were expressed in the RGC-5 cells and in cDNA from human brain and optic nerve, with the exception of stathmin-like 4, which was not expressed in the RGC-5 cells. The patients with PXFG had increased anti-C6orf129 IgG immunoreactivity compared with that in the control subjects (P < 0.05). CONCLUSIONS: Screening high-density protein arrays identifies unique antibody profiles that may discriminate between patients with and without PXFG. Characterization of the autoantibody repertoire may provide new insights into the pathophysiology of PXFG.

Helicobacter pylori IgG antibodies in aqueous humor and serum of subjects with primary open angle and pseudo-exfoliation glaucoma in a South Indian population.

PURPOSE: A prospective, nonrandomized, comparative study was carried out to investigate levels of anti-Helicobacter pylori-specific IgG antibodies in the aqueous humor and serum of patients with primary open angle glaucoma (POAG), exfoliation syndrome [pseudo-exfoliation glaucoma (PXFG)], and with normotensive cataract patients, who served as controls. METHODS: Aqueous humor was aspirated at the beginning of glaucoma surgery from 50 eyes of 50 patients with POAG, with PXFG and at the beginning of phacoemulsification cataract surgery from controls. Serum samples were obtained. Anti-H. pylori IgG concentration in the aqueous humor and serum was measured by means of enzyme linked immunosorbent assay. RESULTS: Serum analysis of anti-H. pylori IgG antibodies revealed statistically significant difference between POAG and PXFG (52.26+/-52.51 vs. 25.22+/-35.27, P=0.01). Also, the difference between POAG and controls was statistically significant (54.05+/-55.04 vs. 33.83+/-41.73, P=0.04). However, on comparing PXFG with the control group, the difference was statistically insignificant (P=0.12). The mean concentration of anti-H. pylori IgG antibodies in aqueous humor of patients in POAG and controls were not statistically different (3.93+/-5.14 vs. 2.65+/-2.87, respectively, P=0.73). The mean concentration of anti-H. pylori IgG antibodies in aqueous humor of patients in PXFG and controls were not statistically different (8.87+/-30.25 vs. 2.65+/-2.87, respectively, P=0.83). There was also no statistical difference of IgG levels between POAG and PXFG (3.93+/-5.14 vs. 8.87+/-30.25, respectively, P=0.87). CONCLUSIONS: The levels of anti-H. pylori IgG titers in sera of individuals with POAG were significantly higher compared with PXFG and control groups. We support the hypothesis of the role of anti-H. pylori antibodies in causative mechanism for POAG. We could not find a significant link between the anti-H. pylori IgG antibodies and the PXFG.

No association between Helicobacter pylori infection or CagA-bearing strains and glaucoma.

BACKGROUND AND PURPOSE: Accumulating evidence indicates that a variety of infections contribute to the pathogenesis of glaucoma. The role of Helicobacter pylori infection in glaucoma is controversial. DESIGN: Prospective, population-based study. PARTICIPANTS: Patients with various types of glaucoma and a control group of patients with cataract. METHODS: We evaluated seropositivity to H. pylori and to its cytotoxin-associated gene A (CagA) product in patients with various types of glaucoma and compared the findings to those of a control group of patients with cataract. RESULTS: H. pylori infection and CagA seropositivity were detected in 31/51 (60.8%) and 26/51 (51%) glaucoma patients compared with 22/36 (61.1%) and 19/36 (52%) control patients, respectively (P=0.88, 0.67, not significant). Similar rates of H. pylori infection and CagA-positive strain were found in all glaucoma subgroups, and none of them was statistically different from those of controls. CONCLUSIONS: Neither H. pylori infection nor seropositivity for virulent CagA-bearing H. pylori strains have significant association with the occurrence of glaucoma of any type.

IgG antibody patterns in aqueous humor of patients with primary open angle glaucoma and pseudoexfoliation glaucoma.

PURPOSE: There has been growing body of evidence indicating an immunological component in the pathogenesis of glaucoma. Several studies found specific antibodies in sera of glaucoma patients. Recently, we detected antibody profiles in aqueous humor of patients with normal tension glaucoma that showed significant differences when compared to control subjects. The aim of this study was to compare the IgG antibody patterns against retinal antigens in aqueous humor of patients with primary open angle glaucoma, pseudoexfoliation glaucoma, and control subjects then to identify some of the important biomarkers of this study. METHODS: Aqueous humor of 44 patients was analyzed: 15 patients with primary open angle glaucoma (POAG), 14 patients with pseudoexfoliation glaucoma (PEX), and 15 control subjects (CO). The aqueous was tested against western blots of bovine retinal antigens. The IgG antibody patterns were analyzed by multivariate statistical techniques. Some of the important biomarkers were identified via mass spectrometry (Maldi-TOFTOF). RESULTS: All patients showed complex patterns of IgG antibodies against retinal antigens. The discriminant analysis revealed a statistically significant difference between the antibody profiles of the POAG and the CO group (p=0.00018). There was also a statistically significant difference between the antibody profiles of the PEX and the CO group (p=0.0013). Not only were up-regulations in the glaucoma groups observed, compared to controls, but also down-regulations. There was no significant difference between the antibody patterns of the POAG and PEX group. The identified biomarkers included heat shock protein 27, alpha-enolase, actin, and GAPDH. CONCLUSIONS: We could show significant differences between the IgG antibody profiles of the glaucoma groups (PEX and POAG) and controls. The analysis of intraocular antibodies could provide further hints for autoimmune involvement in glaucoma.

[Pseudo-exfoliative syndrome--etiology, clinical aspects, diagnosis]. / Sindromul Pseudoexfoliativ-- etiopatogenie, clinica, diagnostic.

The pseudoexfoliative syndrome is a systemic anomaly that primarily affects the eye. Many etiopathogenic mechanisms were found using ELISA, PCR and western immunoblot. The presence of the pseudoexfoliative material causes important changes to the cornea, camerular angle, lens and zonules. Cataract surgery in pseudoexfoliative syndrome is difficult. Pseudoexfoliative glaucoma is aggressive, with great pressure differences between the two eyes, also with important circadian variations of intraocular pressure and has a reserved prognostic.

Increased levels of Helicobacter pylori IgG antibodies in aqueous humor of patients with primary open-angle and exfoliation glaucoma.

PURPOSE: A prospective, nonrandomized, comparative study was carried out to investigate the levels of anti- H. pylori-specific IgG antibodies in the aqueous humor and serum of patients with primary open-angle glaucoma (POAG) and patients with exfoliation glaucoma (XFG), and to compare them with those in age-matched cataract patients. METHODS: Aqueous humor was aspirated at the beginning of glaucoma surgery from 26 eyes of 26 patients with POAG, 27 eyes of 27 patients with XFG, and at the beginning of phakoemulsification cataract surgery from 31 eyes of 31 age-matched normotensive cataract patients. Serum samples were obtained the day before surgery from all patients. Anti- H. pylori IgG concentration in the aqueous humor and serum was measured by means of an enzyme-linked immunosorbent assay. RESULTS: The mean concentration (+/- SE) of anti- H. pylori-specific IgG was significantly greater in the aqueous humor samples from patients with POAG (14.27+/-3.86 U/ml) and XFG (14.25+/-3.39 U/ml) than in those from age-matched control cataract patients (4.67+/-1.07 U/ml); ( P=0.006 and P=0.003, respectively). No difference was observed in the levels of H. pylori-specific IgG antibodies between POAG and XFG aqueous humor samples ( P=0.5). Similarly, the serum concentration of anti- H. pylori antibodies was significantly greater in patients with POAG (69.96+/-9.69 U/ml; P=0.013) and XFG (81.37+/-10.62 U/ml; P=0.002) than in the cataract controls (44.16+/-6.48 U/ml). The mean vertical cupping correlated significantly with the titer of anti- H. pylori-specific IgG in the aqueous humor in the POAG patients. CONCLUSION: H. pylori-specific IgG antibody levels are significantly increased in the aqueous humor and serum of patients with POAG and XFG. Moreover, the titer of anti- H. pylori antibody in the aqueous humor might reflect the severity of glaucomatous damage in POAG patients. These findings support a role for H. pylori infection in the pathobiology of these two common glaucomas.

Kynurenine and neopterin in the aqueous humor of the anterior chamber of the eye and in serum of cataract patients.

Serum kynurenine, neopterin, selenium concentrations; neopterin concentrations in aqueous humour from anterior chamber of eye and selenium content in lenses were determined in 40 catarat patients with and in 51--without pseudoexfoliation (PES). Significantly increased kynurenine and neopterin levels in serum and neopterin concentration in aqueous humour were observed in mature cataract patients with PES compared to whose without. These patients also presented with the lowest content of selenium in serum and lens, compared with cataract patients without PES. Increased concentrations of neopterin in serum and aqueous humour of anterior chamber of eyes suggest an increased degree of oxidative stress in patients with PES. Thus, the results support the role of oxidative stress in the development of PES in cataract patients. The decreased content of selenium may elicit immune system activation via an increased oxidative stress as it is indicated by the increased formation of kynurenine and neopterin.

Immunoelectron microscopic localization of the HNK-1 carbohydrate epitope in the anterior segment of pseudoexfoliation and normal eyes.

PURPOSE: To study the presence of the cell-adhesion related HNK-1 carbohydrate epitope in the anterior segment of pseudoexfoliation and normal eyes by immunoelectron microscopy. METHODS: Anterior segment tissue of 6 autopsy eyes with pseudoexfoliation (PEX) syndrome (5 eyes without glaucoma and 1 with glaucoma), and 6 normal autopsy eyes without PEX syndrome were studied by an electron microscopic immunogold technique using a monoclonal antibody to the HNK-1 epitope. RESULTS: In both normal and PEX eyes, the HNK-1 epitope could be immunolocalized to the basement membranes of both ciliary epithelia and posterior iris pigmented epithelium, to the lens capsule and zonular lamella, and to the pigmented epithelial cells of iris and ciliary body. Within the inner connective tissue layer of the ciliary body, the gold label was mainly associated with the periphery of elastic fibers and microfibrillar bundles. PEX material on the surfaces of posterior iris, ciliary body, anterior lens capsule, zonular fibers, and uveal part of the trabecular meshwork reacted strongly with the HNK-1 antibody. In contrast, PEX material accumulations within the iris stroma or the juxtacanalicular tissue of the trabecular meshwork showed only weak immunoreactivity, while PEX material in the conjunctiva was totally negative. CONCLUSIONS: The wide distribution of the HNK-1 epitope in anterior segment tissues and its association with a variety of extracellular and cellular structures was ultrastructurally demonstrated. In PEX syndrome, the varying labelling density of PEX fibers indicates a deviating carbohydrate composition in different locations of the eye. The HNK-1 epitope might be involved in the adhesiveness of PEX deposits on intraocular surfaces.

Increased IgA antibodies to gluten and gliadin in serum of persons with ocular pseudo-exfoliation.

Three of 12 persons with pseudo-exfoliation syndrome revealed increased IgA serum antibodies to gluten and gliadin vs none in the control group. The phenomenon is suggested to indicate barrier breakdown in the gut wall.

The HNK-1 epitope in the inner connective tissue layer of the human ciliary body in exfoliation syndrome and various types of glaucoma.

Possible changes in the expression of the HNK-1 carbohydrate epitope in the inner connective tissue layer of the human ciliary body, located between the ciliary epithelium and muscle, was studied using 2 formalin-fixed, paraffin-embedded eyes with exfoliation syndrome, 33 eyes with different types of glaucoma, and 21 morphologically normal control eyes. A strong immunoreaction delineating cell process was observed in this layer with monoclonal antibodies HNK-1 and VC1.1 recognizing the HNK-1 epitope in control specimens, whereas partly granular immunoreaction was present in eyes with exfoliation syndrome. Exfoliation material was also immunoreactive. In all types of advanced glaucoma, the immunoreaction was mostly granular in nature and greatly diminished. No difference in HNK-1 immunoreactivity between control and glaucoma eyes was seen in the retina and ciliary epithelium. Elevated intraocular pressure, either directly or by decreasing blood flow to the ciliary body, may cause degenerative or metabolic changes in the inner connective tissue layer cells that bear or secrete molecules sharing the HNK-1 epitope. The partly granular immunoreactivity in eyes with exfoliation syndrome only indicates changes in this epitope even without an increase in intraocular pressure.

Immunoreactivity of exfoliation material for the cell adhesion-related HNK-1 carbohydrate epitope.

OBJECTIVE: To study the presence of the HNK-1 epitope in exfoliation material. METHODS: Twenty-six formalin-fixed, paraffin-embedded human eyes with exfoliation syndrome and 30 control eyes were studied immunohistochemically with monoclonal antibodies HNK-1 and VC1.1 to the HNK-1 epitope. RESULTS: Exfoliation material reacted consistently with antibodies to the HNK-1 epitope. The zonular lamella of the lens, inner surface of the nonpigmented ciliary epithelium, and inner connective tissue layer of the ciliary body were also labeled, but the lens capsule, epithelium, and zonules were not immunoreactive. Several blood vessels of the iris showed granular immunoreaction beneath the endothelium in all exfoliation eyes and in 11 (37%) of 30 control eyes, representing older age groups. CONCLUSIONS: The zonular lamella, nonpigmented ciliary epithelium, or the inner connective tissue layer may be responsible for the HNK-1 epitope in exfoliation material. Since this epitope is shared by many cell-adhesion molecules, its presence in exfoliation material might be of pathogenetic significance to the formation of the deposits.